Vaccines containing peptides linked to stress proteins are the most potent known T cell immunogens. However, the precise mechanisms by which they activate immune cells remain unclear. To address this, we will track activated dendritic cells (DCs) and CD8+ T cells in mice immunized with antigens fused to the stress protein hsp70, and determine which cytokines and cell surface receptors are required for their responses. Our preliminary studies reveal hspTO fusion vaccines elicit much more potent and durable specific CD8+ T cell responses than antigen alone or with other adjuvants. Our underlying hypothesis is that antigen/hsp70 fusions are potent vaccines because they stimulate DCs to provide an optimal microenvironment for CD8+ T cell activation. This microenvironment is defined by high antigen density and strong costimulation. To test this, we will track in vivo DC and CD8+ T cell responses in draining lymph nodes, and the resultant effector and memory CD8+ T lymphocytes in the periphery. Novel aspects of our proposal include: 1) quantifying the amount of antigen presented by activated DCs, 2) the effect hsp70 fusion proteins have on DC lifespan, 3) determining which of the several reported hsp70-receptors are necessary or sufficient for delivering activation signals to DCs in vivo, and 4) determining the costimulatory and cytokines required for the profound effect our vaccine has CD8+ T cell memory. The results of these experiments will allow us to manipulate stress protein-induced responses rationally with the ultimate goal of maximizing their use for cancer therapy.